Novel Antimicrobial Approaches to Combat Bacterial Biofilms Associated with Urinary Tract Infections.

Urinary tract infections (UTIs) are prevalent bacterial infections in both community and healthcare settings. They account for approximately 40% of all bacterial infections and require around 15% of all antibiotic prescriptions. Although antibiotics have traditionally been used to treat UTIs for several decades, the significant increase in antibiotic resistance in recent years has made many previously effective treatments ineffective. Biofilm on medical equipment in healthcare settings creates a reservoir of pathogens that can easily be transmitted to patients. Urinary catheter infections are frequently observed in hospitals and are caused by microbes that form a biofilm after a catheter is inserted into the bladder. Managing infections caused by biofilms is challenging due to the emergence of antibiotic resistance. Biofilms enable pathogens to evade the host's innate immune defences, resulting in long-term persistence. The incidence of sepsis caused by UTIs that have spread to the bloodstream is increasing, and drug-resistant infections may be even more prevalent. While the availability of upcoming tests to identify the bacterial cause of infection and its resistance spectrum is critical, it alone will not solve the problem; innovative treatment approaches are also needed. This review analyses the main characteristics of biofilm formation and drug resistance in recurrent uropathogen-induced UTIs. The importance of innovative and alternative therapies for combatting biofilm-caused UTI is emphasised.

The Challenge of Overcoming Antibiotic Resistance in Carbapenem-Resistant Gram-Negative Bacteria: "Attack on Titan".

The global burden of bacterial resistance remains one of the most serious public health concerns. Infections caused by multidrug-resistant (MDR) bacteria in critically ill patients require immediate empirical treatment, which may not only be ineffective due to the resistance of MDR bacteria to multiple classes of antibiotics, but may also contribute to the selection and spread of antimicrobial resistance. Both the WHO and the ECDC consider carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Acinetobacter baumannii (CRAB) to be the highest priority. The ability to form biofilm and the acquisition of multiple drug resistance genes, in particular to carbapenems, have made these pathogens particularly difficult to treat. They are a growing cause of healthcare-associated infections and a significant threat to public health, associated with a high mortality rate. Moreover, co-colonization with these pathogens in critically ill patients was found to be a significant predictor for in-hospital mortality. Importantly, they have the potential to spread resistance using mobile genetic elements. Given the current situation, it is clear that finding new ways to combat antimicrobial resistance can no longer be delayed. The aim of this review was to evaluate the literature on how these pathogens contribute to the global burden of AMR. The review also highlights the importance of the rational use of antibiotics and the need to implement antimicrobial stewardship principles to prevent the transmission of drug-resistant organisms in healthcare settings. Finally, the review discusses the advantages and limitations of alternative therapies for the treatment of infections caused by these "titans" of antibiotic resistance.

Urinary Tract Infections: The Current Scenario and Future Prospects.

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, occurring in both community and healthcare settings. Although the clinical symptoms of UTIs are heterogeneous and range from uncomplicated (uUTIs) to complicated (cUTIs), most UTIs are usually treated empirically. Bacteria are the main causative agents of these infections, although more rarely, other microorganisms, such as fungi and some viruses, have been reported to be responsible for UTIs. Uropathogenic Escherichia coli (UPEC) is the most common causative agent for both uUTIs and cUTIs, followed by other pathogenic microorganisms, such as Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, and Staphylococcus spp. In addition, the incidence of UTIs caused by multidrug resistance (MDR) is increasing, resulting in a significant increase in the spread of antibiotic resistance and the economic burden of these infections. Here, we discuss the various factors associated with UTIs, including the mechanisms of pathogenicity related to the bacteria that cause UTIs and the emergence of increasing resistance in UTI pathogens.

SARS-CoV-2 Infection in Patients with Cystic Fibrosis: What We Know So Far.

Respiratory infections are the most common and most frequent diseases, especially in children and the elderly, characterized by a clear seasonality and with an incidence that usually tends to decrease with increasing age. These infections often resolve spontaneously, usually without the need for antibiotic treatment and/or with the possible use of symptomatic treatments aimed at reducing overproduction of mucus and decreasing coughing. However, when these infections occur in patients with weakened immune systems and/or underlying health conditions, their impact can become dramatic and in some cases life threatening. The rapid worldwide spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has caused concern for everyone, becoming especially important for individuals with underlying lung diseases, such as CF patients, who have always paid close attention to implementing protective strategies to avoid infection. However, adult and pediatric CF patients contract coronavirus infection like everyone else. In addition, although numerous studies were published during the first wave of the pandemic on the risk for patients with cystic fibrosis (CF) to develop severe manifestations when infected with SARS-CoV-2, to date, a high risk has been found only for patients with poorer lung function and post-transplant status. In terms of preventive measures, vaccination remains key. The best protection for these patients is to strengthen preventive measures, such as social distancing and the use of masks. In this review, we aim to summarize and discuss recent advances in understanding the susceptibility of CF individuals to SARS-CoV-2 infection.

Recent Advances in the Use of Molecular Methods for the Diagnosis of Bacterial Infections.

Infections caused by bacteria have a major impact on public health-related morbidity and mortality. Despite major advances in the prevention and treatment of bacterial infections, the latter continue to represent a significant economic and social burden worldwide. The WHO compiled a list of six highly virulent multidrug-resistant bacteria named ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) responsible for life-threatening diseases. Taken together with Clostridioides difficile, Escherichia coli, Campylobacter spp., (C. jejuni and C. coli), Legionella spp., Salmonella spp., and Neisseria gonorrhoeae, all of these microorganisms are the leading causes of nosocomial infections. The rapid and accurate detection of these pathogens is not only important for the early initiation of appropriate antibiotic therapy, but also for resolving outbreaks and minimizing subsequent antimicrobial resistance. The need for ever-improving molecular diagnostic techniques is also of fundamental importance for improving epidemiological surveillance of bacterial infections. In this review, we aim to discuss the recent advances on the use of molecular techniques based on genomic and proteomic approaches for the diagnosis of bacterial infections. The advantages and limitations of each of the techniques considered are also discussed.

Extended-spectrum ß-lactamase & carbapenemase-producing fermentative Gram-negative bacilli in clinical isolates from a University Hospital in Southern Italy.

The aim of this study was to determine the prevalence of extended-spectrum ß-lactamases (ESBLs)- and carbapenemase-producing fermentative Gram-negative bacteria (FGNB) in a University Hospital in Southern Italy. These bacteria have the potential to disseminate bacterial resistance in healthcare settings and cause untreatable and prolonged infections associated with high rates of mortality. A retrospective observational study was carried out in a University Hospital in Sicily from January to December 2019. A total of 1046 FGNB were recovered from different clinical samples among which 40%, 15% and 37% were, respectively, MDR, carbapenemase and ESBL producers. Antibiotic resistance profile of FGNB against the first-line drugs was remarkably high. K. pneumoniae (57%) followed by E. coli (27%) were found here as the major sources of ESBL producers. The highest proportion of ESBL producers was from ICU ward (72%), and were isolated from urine samples (63.6%) followed by blood samples (54%). Carbapenemase production among the FGNB in our study was about 0.9%, which is more than twice than the prevalence rate reported by the European Antimicrobial Resistance Surveillance Network (ECDC) (0.4%). To our knowledge, this is the first report on the prevalence of ESBL and carbapenemase-producing FGNB in this region. Our data clearly indicate the importance of implementing antibiotic stewardship strategies in our region to reduce the unnecessary use of antibiotics.

Characterization of an immunogenic cellulase secreted by Cryptococcus pathogens.

Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

Neutrophils Enhance Their Own Influx to Sites of Bacterial Infection via Endosomal TLR-Dependent Cxcl2 Production.

The influx of neutrophils to infection sites is a fundamental step in host defenses against the frequent human pathogen group B Streptococcus (GBS) and other extracellular bacteria. Using a mouse model of GBS-induced peritonitis, we show in this study that the chemokines Cxcl1 and Cxcl2 play distinctive roles in enhancing the recruitment and the antibacterial activities of neutrophils in a manner that is linked to differences in the cellular sources of these mediators. Cell depletion experiments demonstrated that neutrophils make a significant contribution to the in vivo production of Cxcl2 but not Cxcl1. In vitro, neutrophils responded weakly to LPS but released high levels of Cxcl2 after stimulation with GBS or other bacteria. Neutrophil-derived Cxcl2 acted in an autocrinous manner to increase its own production and to enhance antibacterial activities, including the release of oxygen radicals. In both neutrophils and macrophages, the production of Cxcl1/2 largely required the presence of functional UNC93B1, a chaperone protein involved in signaling by endosomal TLRs. Moreover, the phenotype of UNC93B1-defective phagocytes could be recapitulated by the simultaneous absence of TLR7, 9, and 13 but not by the absence of individual TLRs. Collectively, our data show that neutrophils recognize Gram-positive and Gram-negative bacteria by means of multiple phagosomal TLRs, resulting in de novo synthesis of Cxcl2, amplification of neutrophil recruitment, and potentiation of their antibacterial activities. These data may be useful to devise alternative therapeutic strategies aimed at enhancing the recruitment and the functional activities of polymorphonuclear leukocytes during infections caused by antibiotic-resistant bacteria.

Antibacterial activities of almond skins on cagA-positive and-negative clinical isolates of Helicobacter pylori.

BACKGROUND: Helicobacter pylori is known to be a gastric pathogen of humans. Eradication regimens for H. pylori infection have some side effects, compliance problems, relapses, and antibiotic resistance. Therefore, the need for alternative therapies for H. pylori infections is of special interest. We have previously shown that polyphenols from almond skins are active against a range of food-borne pathogens. The aim of this study was to evaluate the antibacterial effects of natural almond skins before and after simulated human digestion and the pure flavonoid compounds epicatechin, naringenin and protocatechuic acid against H. pylori. RESULTS: H. pylori strains were isolated from gastric biopsy samples following standard microbiology procedures. Also, cagA and vacA genes were identified using PCR. Susceptibility studies on 34 strains of H. pylori, including two reference strains (ATCC 43504, ATCC 49503), were performed by the standard agar dilution method. CONCLUSIONS: Polyphenols from almond skins were effective in vitro against H. pylori, irrespective of genotype status and could therefore be used in combination with antibiotics as a novel strategy for antibiotic resistance.

Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria.

It is known that host cells can produce type I IFNs (IFN-alphabeta) after exposure to conserved bacterial products, but the functional consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-alphabeta signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli. In response to GBS challenge, most mice lacking either the IFN-alphabetaR or IFN-beta died from unrestrained bacteremia, whereas all wild-type controls survived. The effect of IFN-alphabetaR deficiency was marked, with mortality surpassing that seen in IFN-gammaR-deficient mice. Animals lacking both IFN-alphabetaR and IFN-gammaR displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-alphabeta was detected in macrophages after exposure to GBS. Moreover, in the absence of IFN-alphabeta signaling, a marked reduction in macrophage production of IFN-gamma, NO, and TNF-alpha was observed after stimulation with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-alphabeta in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat bacterial infections.

Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae.

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.